Cell survival, division, and differentiation are dependent on a delicate balance between the rates of protein synthesis and degradation. The Ubiquitin-Proteasome System (UPS) is one of the primary pathways for regulating protein turnover in the eukaryotic cell through a complex mechanism of enzymes and substrate-recognition proteins. The target of this research, E2- 25K, is a protein component of this system, and has been shown to play a role in neurodegenerative diseases, particularly in diseases of protein misfolding such as Alzheimer's Disease and Huntington's Disease. E2-25K functions to covalently link the small tag protein ubiquitin to other protein substrates destined for degradation by the proteasome. E2-25K interacts with a large number of E3 ligase proteins which help determine specificity and facilitate binding to the substrate. Additionally it has a unique domain which interacts with ubiquitin non-covalently. The long-term objective of this research is to understand the mechanisms for its adaptation to various binding partners and polyubiquitin chain synthesis. The Specific Aims for this application are: 1. Structural and functional characterization of human E2-25K. 2. Identify specific residues of E2-25K involved in binding four known RING finger E3 ligase partners. 3. Identify specific residues of the E2-25K UBA domain involved in binding to di-ubiquitin. 4. Investigate whether oligomerization of E2-25K occurs under certain conditions. The structure of human E2-25K will be examined using both x-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy will also be used to map the relevant binding surfaces of human E2-25K required for interaction with multiple RING (Really Interesting New Gene) finger E3 ubiquitin-ligase partners and with various forms of ubiquitin. The formation of E2-25K oligomers under a variety of conditions will be investigated by size-exclusion chromatography. The results will be used to help define the manner in which E2-25K adapts to multiple pathways, and ultimately to determine the mechanism of polyubiquitin chain formation. PUBLIC HEALTH RELEVANCE: The ability of the cell to degrade its own proteins at the proper time is essential to its development and survival. The target of this research, E2-25K, is a protein component of this cellular degradation system, and has been shown to play a role specifically in both Huntington's Disease (HD) and Alzheimer's Disease (AD). Because E2-25K is implicated in the development of some neurodegenerative diseases, it is an interesting target for drug therapy. This aim of this research is to understand how this protein functions in the cell.